The immune response is a mechanism by which the body defends itself against foreign substances that invade it, causing infection or disease. This mechanism is based on the ability of antibodies produced or administered to the host to bind the antigen though its variable region. Once the antigen is bound by the antibody, the antigen is targeted for destruction, often mediated in part, by the constant region or Fc domain of the antibody.
For example, one activity of the Fc domain of the antibody is to bind complement proteins which can assist in lysing the target antigen, for example, a cellular pathogen. Another activity of the Fc region is to bind to Fc receptors (FcR) on the surface of immune cells, or so-called effector cells, which have the ability to trigger other immune effects. These immune effects include, for example, release of immune activators, regulation of antibody production, endocytosis, phagocytosis, and cell killing. In some clinical applications these responses are crucial for the efficacy of the antibody while in other cases they provoke unwanted side effects. One example of an effector-mediated side effect is the release of inflammatory cytokines causing an acute fever reaction. Another example is the long term deletion of antigen-bearing cells.
The effector function of an antibody can be avoided by using antibody fragments lacking the Fc region (e.g., such as a Fab, Fab′2, or single chain antibody (sFv)) however these fragments have a reduced half-life, only one antigen binding site instead of two (e.g., in the case of Fab antibody fragments and single chain antibodies (sFv)), and are more difficult to purify.
Currently there are limited ways to reduce the effector function of an antibody while retaining the other valuable attributes of the Fc region. One approach is to mutate amino acids on the surface of the antibody that are involved in the effector binding interactions. While some mutations lead to a reduction of effector function, residual activity usually remains. Moreover, these added mutations can make the antibody immunogenic.
Another approach to reduce effector function is to remove sugars that are linked to particular residues in the Fc region, by for example, deleting or altering the residue the sugar is attached to, removing the sugars enzymatically, by producing the antibody in cells cultured in the presence of a glycosylation inhibitor, or by expressing the antibody in cells unable to glycosylate proteins. However, the forgoing approaches leave residual effector function both in the form of complement-dependent cytolytic activity and Fc receptor binding. Thus, a further decrease in effector function would be important to guarantee complete ablation of activity.
Accordingly, a need exists for an improved method of making aglycosylated antibodies with altered or reduced effector function.